Vertebrate skeletal muscle relaxes when CA++ is removed from troponin-tropomyosin a complex of proteins on actin. We previously showed, using singlehead myosin subfragment-1 (S-1) that this inhibition results from a decreased rate of a step in the ATP hydrolysis cycle rather than from sterically blocking the binding of S-1 to actin. We now find that the two-headed fragment of myosin, heavy meromyosin behaves similarly. These studies suggest that myosin may be attached to actin in relaxed muscle. In fact, our stiffness measurements of muscle fibers are consistent with attached myosin in relaxed muscle at 5C, 20 mM ionic strength. Therefore, even in muscle fibers, troponin-tropomyosin can cause relaxation without preventing the binding of myosin to actin. These studies have also been extended to smooth muscle. Chicken gizzard muscle is relaxed by dephosphorylation of myosin. We find that this dephosphorylation acts by blocking a step in the ATPase cycle rather than by blocking binding of the myosin of actin. Thus, in both smooth and skeletal muscle, relaxation can occur without preventing the binding of myosin to actin.